Confocal Microscopy List

FocalCheck test slide

Classic

List

Threaded

13 messages Options

MICROSCOPIA CONFOCAL y CCD

FocalCheck test slide

Does anybody have experience with the FocalCheck
Fluorescence Microscope Test Slides from Molecular Probes.?
I bought Test Slide #1 and I'd focus problems
especially with oil immersion objectives in
different microscopes. They have sent me another
one and I'm having the same trouble focusing.I do
not understand what is happening. ¿Someone has had problems with this kit?

Thanks in advance

M. Teresa

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Oscar Hidalgo Blanco
Amadeo Cazaña Soto
[hidden email]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32

Ignatius, Mike-2

Re: FocalCheck test slide

Dr. Teresa,

The most likely issue is the thickness of the mountant. We place our beads on a glass slide, then overlay with mountant and a 1.5 coverslip (160 to 190 microns thick). If you have a high mag objective with short working distance (WD) and no correction collar, you indeed may not be able to focus deeply enough to see them. There is a wonderful tutorial on this by Dr. Davidson at http://www.microscopyu.com/tutorials/java/aberrations/correctioncollar/

One quick check is to image them at lower magnification - if you can see them, the mountant thickness and short WD are the most likely culprits. (There can also be some variation to coverslip thickness.) Many of the newer objectives have improved WDs at high magnification and numerical aperture and come with correction collars (at a price though).

We are instituting QC and manufacturing controls to prevent our contribution to this issue going forward. You may want to consider buying and mounting the desired stand alone beads directly onto a coverslip for the immediate future (cheaper than a new objective!). We will of course refund your money for the prepared slides if we are unable to resolve the issue with your current equipment.

Kind Regards,

Mike Ignatius

Product Development Manager
Molecular Probes/LifeTechnologies
[hidden email]
541 335-0414

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MICROSCOPIA CONFOCAL y CCD
Sent: Wednesday, March 24, 2010 9:06 AM
To: [hidden email]
Subject: FocalCheck test slide

Does anybody have experience with the FocalCheck
Fluorescence Microscope Test Slides from Molecular Probes.?
I bought Test Slide #1 and I'd focus problems
especially with oil immersion objectives in
different microscopes. They have sent me another
one and I'm having the same trouble focusing.I do
not understand what is happening. ¿Someone has had problems with this kit?

Thanks in advance

M. Teresa

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Oscar Hidalgo Blanco
Amadeo Cazaña Soto
[hidden email]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32

Axel Kurt Preuss

Re: FocalCheck test slide

In reply to this post by MICROSCOPIA CONFOCAL y CCD

You have to put the slide upside down when using inverted

Hope it helps

Axel

Axel K Preuss PhD, IMCB-Central Imaging Facility
Sent from. +65 9271 5622

On Mar 25, 2010, at 12:24 AM, "MICROSCOPIA CONFOCAL y CCD" <[hidden email]
> wrote:

> Does anybody have experience with the FocalCheck
> Fluorescence Microscope Test Slides from Molecular Probes.?
> I bought Test Slide #1 and I'd focus problems
> especially with oil immersion objectives in
> different microscopes. They have sent me another
> one and I'm having the same trouble focusing.I do
> not understand what is happening. ¿Someone has had problems with thi
> s kit?
>
> Thanks in advance
>
> M. Teresa
>
> SERVICIO de MICROSCOPIA CONFOCAL y CCD
> Mª Teresa Seisdedos Domínguez
> Oscar Hidalgo Blanco
> Amadeo Cazaña Soto
> [hidden email]
> Centro Investigaciones Biológicas (CIB) CSIC
> C/Ramiro de Maeztu, 9
> 28040 Madrid
> Phone: + 34-91 8373112 ext.4401
> Fax: + 34-91 536 04 32

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

MICROSCOPIA CONFOCAL y CCD

Re: FocalCheck test slide

>Thanks a lot for your answers.

>Of course I put the slide upside down when using
>inverted microscope and I asked for Molecular
>probes about coverslip and they answered me:
>"The coverslip thickness for the FocalCheck
>prepared slides is 0.13-0.16 mm" It´s no ideal
>but I think that this is not the problem.

M. Teresa

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Oscar Hidalgo Blanco
Amadeo Cazaña Soto
[hidden email]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32

Guy Cox-2

Re: FocalCheck test slide

No, no, it IS the problem! As Mike Ignatius explained, MP put the beads on the slide, not the coverslip, then add mountant and then the coverslip. If you are using a #1.5 coverslip you need to put the beads directly on the coverslip. With the beads on the slide the extra thickness of the mountant needs to be corrected for by using a thinner coverslip - #1 or #0 - which must be found by trial and error. (But since I guess they are using very reproducible conditions they only need to do the test once). At least it seems they have realized they have a problem.

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MICROSCOPIA CONFOCAL y CCD
Sent: Thursday, 25 March 2010 10:15 PM
To: [hidden email]
Subject: Re: FocalCheck test slide

>Thanks a lot for your answers.

>Of course I put the slide upside down when using
>inverted microscope and I asked for Molecular
>probes about coverslip and they answered me:
>"The coverslip thickness for the FocalCheck
>prepared slides is 0.13-0.16 mm" It´s no ideal
>but I think that this is not the problem.

M. Teresa

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Oscar Hidalgo Blanco
Amadeo Cazaña Soto
[hidden email]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32

Peter Gabriel Pitrone

Re: FocalCheck test slide

They sell these "test slides" for hundreds of dollars, yet they don't take the time to make them right?!?! They should be made with correct coverslips chosen for their thickness, deposited on them and then mounted on the glass slide... I see that there is a disconnect some where, how much does it cost them to produce these slides? 10 bucks maybe...

Pete

On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:

> No, no, it IS the problem! As Mike Ignatius explained, MP put the beads on the slide, not the coverslip, then add mountant and then the coverslip. If you are using a #1.5 coverslip you need to put the beads directly on the coverslip. With the beads on the slide the extra thickness of the mountant needs to be corrected for by using a thinner coverslip - #1 or #0 - which must be found by trial and error. (But since I guess they are using very reproducible conditions they only need to do the test once). At least it seems they have realized they have a problem.
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MICROSCOPIA CONFOCAL y CCD
> Sent: Thursday, 25 March 2010 10:15 PM
> To: [hidden email]
> Subject: Re: FocalCheck test slide
>
>> Thanks a lot for your answers.
>
>> Of course I put the slide upside down when using
>> inverted microscope and I asked for Molecular
>> probes about coverslip and they answered me:
>> "The coverslip thickness for the FocalCheck
>> prepared slides is 0.13-0.16 mm" It´s no ideal
>> but I think that this is not the problem.
>
> M. Teresa
>
> SERVICIO de MICROSCOPIA CONFOCAL y CCD
> Mª Teresa Seisdedos Domínguez
> Oscar Hidalgo Blanco
> Amadeo Cazaña Soto
> [hidden email]
> Centro Investigaciones Biológicas (CIB) CSIC
> C/Ramiro de Maeztu, 9
> 28040 Madrid
> Phone: + 34-91 8373112 ext.4401
> Fax: + 34-91 536 04 32

Katharina Thomsen

Re: FocalCheck test slide

In reply to this post by MICROSCOPIA CONFOCAL y CCD

Dear Teresa,

Because we also wanted to place cover slips with a different thickness,
we decided to buy the FocalCheck solution. In principal this should work
in your case, as well. You can just dry them on the slide together with
the cover slip. Unfortunatly, in this configuration they don't last a
long time. Some of the FocalChecks got destroyed, when I tried to mount
them for a longer time. This was the case when I first dried them (~
5µl) for ~ 1 h on the coverslip and than added mounting medium (dako,
fluorescence mounting media). The drying time is then critical. I didn't
like the fluorescence mounting media of molecular probes, because the
probes haven't been strongly fixed, but maybe it works better with the
FocalChecks.

cheers,

Katharina

MICROSCOPIA CONFOCAL y CCD schrieb:

> Does anybody have experience with the FocalCheck Fluorescence
> Microscope Test Slides from Molecular Probes.?
> I bought Test Slide #1 and I'd focus problems especially with oil
> immersion objectives in different microscopes. They have sent me
> another one and I'm having the same trouble focusing.I do not
> understand what is happening. ¿Someone has had problems with this kit?
>
> Thanks in advance
>
> M. Teresa
>
> SERVICIO de MICROSCOPIA CONFOCAL y CCD
> Mª Teresa Seisdedos Domínguez
> Oscar Hidalgo Blanco
> Amadeo Cazaña Soto
> [hidden email]
> Centro Investigaciones Biológicas (CIB) CSIC
> C/Ramiro de Maeztu, 9
> 28040 Madrid
> Phone: + 34-91 8373112 ext.4401
> Fax: + 34-91 536 04 32

Neeraj Gohad-2

Re: FocalCheck test slide

We do the same thing as Katrina mentioned. We bought Focal Check as well as the sub-resolution beads for finding out PSF and made and our own slides. The focal check and the subresolution beads come with protocols for making slides (http://probes.invitrogen.com/media/pis/mp07234.pdf ). You need a very little quantity to make slides, I dilute the beads before spotting them on the coverglass, I prepared my slides as per the protocol and they lasted for a long time.

No commercial interest!

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

Website: http://www.clemson.edu/okeanos

Please note my new email address: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Katharina Thomsen
Sent: Friday, March 26, 2010 12:18 PM
To: [hidden email]
Subject: Re: FocalCheck test slide

Dear Teresa,

Because we also wanted to place cover slips with a different thickness,
we decided to buy the FocalCheck solution. In principal this should work
in your case, as well. You can just dry them on the slide together with
the cover slip. Unfortunatly, in this configuration they don't last a
long time. Some of the FocalChecks got destroyed, when I tried to mount
them for a longer time. This was the case when I first dried them (~
5µl) for ~ 1 h on the coverslip and than added mounting medium (dako,
fluorescence mounting media). The drying time is then critical. I didn't
like the fluorescence mounting media of molecular probes, because the
probes haven't been strongly fixed, but maybe it works better with the
FocalChecks.

cheers,

Katharina

MICROSCOPIA CONFOCAL y CCD schrieb:

Glen MacDonald-2

Re: FocalCheck test slide

It is much easier to find the surface on which the subresolution beads are mounted if a few larger beads of 6 um or 15 um beads are added to the diluted suspension.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

On Mar 26, 2010, at 11:05 AM, Neeraj Gohad wrote:

> We do the same thing as Katrina mentioned. We bought Focal Check as well as the sub-resolution beads for finding out PSF and made and our own slides. The focal check and the subresolution beads come with protocols for making slides (http://probes.invitrogen.com/media/pis/mp07234.pdf ). You need a very little quantity to make slides, I dilute the beads before spotting them on the coverglass, I prepared my slides as per the protocol and they lasted for a long time.
>
> No commercial interest!
>
>
> Best,
>
> Neeraj.
>
> Neeraj V. Gohad, Ph.D.
> Postdoctoral Fellow
> Okeanos Research Group
> Department of Biological Sciences
> 132 Long Hall
> Clemson University
> Clemson,SC-29634
> Phone: 864-656-3597
> Fax: 864-656-0435
>
> Website: http://www.clemson.edu/okeanos
>
> Please note my new email address: [hidden email]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Katharina Thomsen
> Sent: Friday, March 26, 2010 12:18 PM
> To: [hidden email]
> Subject: Re: FocalCheck test slide
>
> Dear Teresa,
>
> Because we also wanted to place cover slips with a different thickness,
> we decided to buy the FocalCheck solution. In principal this should work
> in your case, as well. You can just dry them on the slide together with
> the cover slip. Unfortunatly, in this configuration they don't last a
> long time. Some of the FocalChecks got destroyed, when I tried to mount
> them for a longer time. This was the case when I first dried them (~
> 5µl) for ~ 1 h on the coverslip and than added mounting medium (dako,
> fluorescence mounting media). The drying time is then critical. I didn't
> like the fluorescence mounting media of molecular probes, because the
> probes haven't been strongly fixed, but maybe it works better with the
> FocalChecks.
>
> cheers,
>
> Katharina
>
>
>
> MICROSCOPIA CONFOCAL y CCD schrieb:
>> Does anybody have experience with the FocalCheck Fluorescence
>> Microscope Test Slides from Molecular Probes.?
>> I bought Test Slide #1 and I'd focus problems especially with oil
>> immersion objectives in different microscopes. They have sent me
>> another one and I'm having the same trouble focusing.I do not
>> understand what is happening. ¿Someone has had problems with this kit?
>>
>> Thanks in advance
>>
>> M. Teresa
>>
>> SERVICIO de MICROSCOPIA CONFOCAL y CCD
>> Mª Teresa Seisdedos Domínguez
>> Oscar Hidalgo Blanco
>> Amadeo Cazaña Soto
>> [hidden email]
>> Centro Investigaciones Biológicas (CIB) CSIC
>> C/Ramiro de Maeztu, 9
>> 28040 Madrid
>> Phone: + 34-91 8373112 ext.4401
>> Fax: + 34-91 536 04 32

Ignatius, Mike-2

Re: FocalCheck test slide

In reply to this post by Peter Gabriel Pitrone

Dear Listservors,

Thank you all very much for your contributions to this discussion.

We have always used #1.5 coverslips(which range from 160 to 190 microns). All biological objectives (except those with correction collars) are designed for these. Going to #1 coverslips would only narrow things by 30 microns at best and defeat the optical correction factor.

We continue to encourage do-it-yourselfers (DYI) slides as others have mentioned, and include protocols to do this. We are very generous in the quantities of beads shipped - you can make dozens to hundreds of slides from one vial. However we developed the multiple bead slides as a cost savings to the customer needing more than one bead type. Buying 10 bead preps is 2-5x what one of the prepared slides will cost.

Unfortunately, the prepared slides are a quite a challenges to make in quantity. We have strict QC standards - our scrap rate and thus costs are high. We haven't implemented a QC step with oil objectives because of the damage to the slide that would entail. As mentioned in an earlier post we are addressing this going forward (first up mounting on coverslips).

We will refund anyone that is unable to see their beads on our slides of course. Just call our Tech Support for a credit or replacement, at 800-955-6288, then selection 2, then selection 4 to request this.

Regards,

Mike Ignatius

Molecular Probes/Lifetechnologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peter Pitrone
Sent: Friday, March 26, 2010 6:03 AM
To: [hidden email]
Subject: Re: FocalCheck test slide

They sell these "test slides" for hundreds of dollars, yet they don't take the time to make them right?!?! They should be made with correct coverslips chosen for their thickness, deposited on them and then mounted on the glass slide... I see that there is a disconnect some where, how much does it cost them to produce these slides? 10 bucks maybe...

Pete

On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:

Guy Cox-2

Re: FocalCheck test slide

Mike,

Obviously mounting the beads on the coverslip will fix the problem, and if your process permits this it is the easiest solution. But there is nothing "wrong" or "incorrect" about thinner coverslips. The magic figure is .17mm at RI 1.515 between the top of the coverslip and the sample. A .17mm coverslip plus a layer of mountant does not meet this criterion, and there will be spherical aberration with a dry lens and possible inability to focus with a short WD oil lens. If your drop of mountant is consistent in volume, and is spread to just reach the edges of the coverslip, it is pretty simple to measure the resulting total thickness and thus which coverslip to use. In my experience, #1 is usually the best fit. (Or #0 for a sample with significant thickness, which is not the case here).

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ignatius, Mike
Sent: Saturday, 27 March 2010 8:31 AM
To: [hidden email]
Subject: Re: FocalCheck test slide

Dear Listservors,

Thank you all very much for your contributions to this discussion.

We have always used #1.5 coverslips(which range from 160 to 190 microns). All biological objectives (except those with correction collars) are designed for these. Going to #1 coverslips would only narrow things by 30 microns at best and defeat the optical correction factor.

We continue to encourage do-it-yourselfers (DYI) slides as others have mentioned, and include protocols to do this. We are very generous in the quantities of beads shipped - you can make dozens to hundreds of slides from one vial. However we developed the multiple bead slides as a cost savings to the customer needing more than one bead type. Buying 10 bead preps is 2-5x what one of the prepared slides will cost.

Unfortunately, the prepared slides are a quite a challenges to make in quantity. We have strict QC standards - our scrap rate and thus costs are high. We haven't implemented a QC step with oil objectives because of the damage to the slide that would entail. As mentioned in an earlier post we are addressing this going forward (first up mounting on coverslips).

We will refund anyone that is unable to see their beads on our slides of course. Just call our Tech Support for a credit or replacement, at 800-955-6288, then selection 2, then selection 4 to request this.

Regards,

Mike Ignatius

Molecular Probes/Lifetechnologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peter Pitrone
Sent: Friday, March 26, 2010 6:03 AM
To: [hidden email]
Subject: Re: FocalCheck test slide

They sell these "test slides" for hundreds of dollars, yet they don't take the time to make them right?!?! They should be made with correct coverslips chosen for their thickness, deposited on them and then mounted on the glass slide... I see that there is a disconnect some where, how much does it cost them to produce these slides? 10 bucks maybe...

Pete

On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:

George McNamara

Re: FocalCheck test slide ... please switch to 170 +/- 0.5 um coverglasses

In reply to this post by Ignatius, Mike-2

Hi Mike and list,

Putting the beads on the coverglass is a good
start, but #1.5 is way too high a tolerance range
for best performing fluorescence microscopes and
nanoscopes. How about switching to the Zeiss 170
um coverglasses or equivalent. $40 for pack of 1,000 pieces, 18x18 mm.

I also encourage mattek
(www.glassbottomdishes.com) and everyone else to switch to 170 um coverglasses.

I also have a negative recommendation with
respect to Lab-Tek chamber slides. I had a
confocal user Friday who had their cells on a
Lab-Tek chamber slides with (unknown thickness)
coverglass applied by the user. While I like the
convenience of the blue ink ridges for finding
the focus, the Lab-Tek slides ridge thickness is
a significant gap between the objective lens and
cells. I also have no idea what kind of
coverglass thickness the user had or their
mounting medium. I also suspect they had left the
slides in a refrigerator, and managed to get
water condensation mixed with immersion oil on
imaging. What I can say is that their image
quality was mediocre, even after cleaned the oil
off the lens and coverglass and reapplied fresh
oil. The Lab-Tek slides also have the wonderful
property of warping during focusing - making it a
waste of time to try to do Z-series.

Sincerely,

George

At 05:31 PM 3/26/2010, you wrote:

>Dear Listservors,
>
>Thank you all very much for your contributions to this discussion.
>
>We have always used #1.5 coverslips(which range
>from 160 to 190 microns). All biological
>objectives (except those with correction
>collars) are designed for these. Going to #1
>coverslips would only narrow things by 30
>microns at best and defeat the optical correction factor.
>
>We continue to encourage do-it-yourselfers (DYI)
>slides as others have mentioned, and include
>protocols to do this. We are very generous in
>the quantities of beads shipped - you can make
>dozens to hundreds of slides from one vial.
>However we developed the multiple bead slides as
>a cost savings to the customer needing more than
>one bead type. Buying 10 bead preps is 2-5x
>what one of the prepared slides will cost.
>
>Unfortunately, the prepared slides are a quite a
>challenges to make in quantity. We have strict
>QC standards - our scrap rate and thus costs are
>high. We haven't implemented a QC step with oil
>objectives because of the damage to the slide
>that would entail. As mentioned in an earlier
>post we are addressing this going forward (first up mounting on coverslips).
>
>We will refund anyone that is unable to see
>their beads on our slides of course. Just call
>our Tech Support for a credit or replacement, at
>800-955-6288, then selection 2, then selection 4 to request this.
>
>Regards,
>
>Mike Ignatius
>
>Molecular Probes/Lifetechnologies
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Peter Pitrone
>Sent: Friday, March 26, 2010 6:03 AM
>To: [hidden email]
>Subject: Re: FocalCheck test slide
>
>They sell these "test slides" for hundreds of
>dollars, yet they don't take the time to make
>them right?!?! They should be made with correct
>coverslips chosen for their thickness, deposited
>on them and then mounted on the glass slide... I
>see that there is a disconnect some where, how
>much does it cost them to produce these slides? 10 bucks maybe...
>
>Pete
>
>On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:
>
> > No, no, it IS the problem! As Mike Ignatius
> explained, MP put the beads on the slide, not
> the coverslip, then add mountant and then the
> coverslip. If you are using a #1.5 coverslip
> you need to put the beads directly on the
> coverslip. With the beads on the slide the
> extra thickness of the mountant needs to be
> corrected for by using a thinner coverslip - #1
> or #0 - which must be found by trial and
> error. (But since I guess they are using very
> reproducible conditions they only need to do
> the test once). At least it seems they have realized they have a problem.
> >
> > Guy
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[hidden email]] On
> Behalf Of MICROSCOPIA CONFOCAL y CCD
> > Sent: Thursday, 25 March 2010 10:15 PM
> > To: [hidden email]
> > Subject: Re: FocalCheck test slide
> >
> >> Thanks a lot for your answers.
> >
> >> Of course I put the slide upside down when using
> >> inverted microscope and I asked for Molecular
> >> probes about coverslip and they answered me:
> >> "The coverslip thickness for the FocalCheck
> >> prepared slides is 0.13-0.16 mm" It´s no ideal
> >> but I think that this is not the problem.
> >
> > M. Teresa
> >
> > SERVICIO de MICROSCOPIA CONFOCAL y CCD
> > Mª Teresa Seisdedos Domínguez
> > Oscar Hidalgo Blanco
> > Amadeo Cazaña Soto
> > [hidden email]
> > Centro Investigaciones Biológicas (CIB) CSIC
> > C/Ramiro de Maeztu, 9
> > 28040 Madrid
> > Phone: + 34-91 8373112 ext.4401
> > Fax: + 34-91 536 04 32

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the
entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

Peter Gabriel Pitrone

Re: FocalCheck test slide ... please switch to 170 +/- 0.5 um coverglasses

Hello George,

You wrote:
"Putting the beads on the coverglass is a good start, but #1.5 is way too
high a tolerance range for best performing fluorescence microscopes and
nanoscopes. How about switching to the Zeiss 170 um coverglasses or
equivalent. $40 for pack of 1,000 pieces, 18x18 mm."

This is exactly what I was implying when I said "They (meaning Molecular
Probes, from here on MP) should be made with correct coverslips chosen for
their thickness", however I didn't want to tell MP who to buy these
coverslips from (to be fair there are other companies making high
precision glass cover slips - Karl. Hecht KG, Sondheim/Rhön, Germany for
example). It is preposterous to me that MP hasn't done so already. We are
talking about high quality "Focal" bead check here. How can one trust the
PSF when the sample was slapped together with what ever was lying around?

Pete

P.S. What is the R.I. of the common coverslip? After a Google search for
another coverslip manufacturer, I came across a paper stating "The ASTM
requires that coverslips have a refractive index of 1.523 + .0052". Is
this true?

> Hi Mike and list,
>
> Putting the beads on the coverglass is a good
> start, but #1.5 is way too high a tolerance range
> for best performing fluorescence microscopes and
> nanoscopes. How about switching to the Zeiss 170
> um coverglasses or equivalent. $40 for pack of 1,000 pieces, 18x18 mm.
>
> I also encourage mattek
> (www.glassbottomdishes.com) and everyone else to switch to 170 um
> coverglasses.
>
> I also have a negative recommendation with
> respect to Lab-Tek chamber slides. I had a
> confocal user Friday who had their cells on a
> Lab-Tek chamber slides with (unknown thickness)
> coverglass applied by the user. While I like the
> convenience of the blue ink ridges for finding
> the focus, the Lab-Tek slides ridge thickness is
> a significant gap between the objective lens and
> cells. I also have no idea what kind of
> coverglass thickness the user had or their
> mounting medium. I also suspect they had left the
> slides in a refrigerator, and managed to get
> water condensation mixed with immersion oil on
> imaging. What I can say is that their image
> quality was mediocre, even after cleaned the oil
> off the lens and coverglass and reapplied fresh
> oil. The Lab-Tek slides also have the wonderful
> property of warping during focusing - making it a
> waste of time to try to do Z-series.
>
>
> Sincerely,
>
> George
>
>
> At 05:31 PM 3/26/2010, you wrote:
>>Dear Listservors,
>>
>>Thank you all very much for your contributions to this discussion.
>>
>>We have always used #1.5 coverslips(which range
>>from 160 to 190 microns). All biological
>>objectives (except those with correction
>>collars) are designed for these. Going to #1
>>coverslips would only narrow things by 30
>>microns at best and defeat the optical correction factor.
>>
>>We continue to encourage do-it-yourselfers (DYI)
>>slides as others have mentioned, and include
>>protocols to do this. We are very generous in
>>the quantities of beads shipped - you can make
>>dozens to hundreds of slides from one vial.
>>However we developed the multiple bead slides as
>>a cost savings to the customer needing more than
>>one bead type. Buying 10 bead preps is 2-5x
>>what one of the prepared slides will cost.
>>
>>Unfortunately, the prepared slides are a quite a
>>challenges to make in quantity. We have strict
>>QC standards - our scrap rate and thus costs are
>>high. We haven't implemented a QC step with oil
>>objectives because of the damage to the slide
>>that would entail. As mentioned in an earlier
>>post we are addressing this going forward (first up mounting on
>> coverslips).
>>
>>We will refund anyone that is unable to see
>>their beads on our slides of course. Just call
>>our Tech Support for a credit or replacement, at
>>800-955-6288, then selection 2, then selection 4 to request this.
>>
>>Regards,
>>
>>Mike Ignatius
>>
>>Molecular Probes/Lifetechnologies
>>
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Peter Pitrone
>>Sent: Friday, March 26, 2010 6:03 AM
>>To: [hidden email]
>>Subject: Re: FocalCheck test slide
>>
>>They sell these "test slides" for hundreds of
>>dollars, yet they don't take the time to make
>>them right?!?! They should be made with correct
>>coverslips chosen for their thickness, deposited
>>on them and then mounted on the glass slide... I
>>see that there is a disconnect some where, how
>>much does it cost them to produce these slides? 10 bucks maybe...
>>
>>Pete
>>
>>On Mar 26, 2010, at 12:25 PM, Guy Cox wrote:
>>
>> > No, no, it IS the problem! As Mike Ignatius
>> explained, MP put the beads on the slide, not
>> the coverslip, then add mountant and then the
>> coverslip. If you are using a #1.5 coverslip
>> you need to put the beads directly on the
>> coverslip. With the beads on the slide the
>> extra thickness of the mountant needs to be
>> corrected for by using a thinner coverslip - #1
>> or #0 - which must be found by trial and
>> error. (But since I guess they are using very
>> reproducible conditions they only need to do
>> the test once). At least it seems they have realized they have a
>> problem.
>> >
>> > Guy
>> >
>> > -----Original Message-----
>> > From: Confocal Microscopy List
>> [mailto:[hidden email]] On
>> Behalf Of MICROSCOPIA CONFOCAL y CCD
>> > Sent: Thursday, 25 March 2010 10:15 PM
>> > To: [hidden email]
>> > Subject: Re: FocalCheck test slide
>> >
>> >> Thanks a lot for your answers.
>> >
>> >> Of course I put the slide upside down when using
>> >> inverted microscope and I asked for Molecular
>> >> probes about coverslip and they answered me:
>> >> "The coverslip thickness for the FocalCheck
>> >> prepared slides is 0.13-0.16 mm" It´s no ideal
>> >> but I think that this is not the problem.
>> >
>> > M. Teresa
>> >
>> > SERVICIO de MICROSCOPIA CONFOCAL y CCD
>> > Mª Teresa Seisdedos Domínguez
>> > Oscar Hidalgo Blanco
>> > Amadeo Cazaña Soto
>> > [hidden email]
>> > Centro Investigaciones Biológicas (CIB) CSIC
>> > C/Ramiro de Maeztu, 9
>> > 28040 Madrid
>> > Phone: + 34-91 8373112 ext.4401
>> > Fax: + 34-91 536 04 32
>
>
>
>
>
>
>
> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
> http://www.sylvester.org/AICF/pubspectra.zip (the
> entire 2000+ spectra .xlsx file is in the zip file)
> http://home.earthlink.net/~geomcnamara
>

Peter Gabriel Pitrone
Light Microscopy Facility
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden